Abstract Rapid advances in DNA sequencing technologies over the past decade have transformed scientists' ability to read the genome, epigenome, and transcriptome of all living organisms, illuminating the genetic architecture and dynamics of biological systems. Establishing causal linkages between observed genetic changes to cellular function, however, requires the ability to control or modulate endogenous genetic sequences or epigenetic states. DNA engineering technologies enable researchers to dissect the function of specific genetic elements or correct disease-causing mutations. However, simple and scalable tools to study and manipulate RNA lag significantly behind their DNA counterparts. The proposed work will focus on developing a heterologous RNA-binding system in eukaryotic cells for the direct and specific perturbation of endogenous transcripts. Analogous to programmable DNA-binding systems such as TAL effectors or CRISPR-Cas9, such RNA targeting technologies would enable diverse applications in addition to RNA knockdown, from translational regulation and allele discrimination, to transcript splicing, trafficking, and visualization. Cas9 nucleases from type II CRISPR systems have been recently implicated in non-canonical gene regulatory functions beyond their prototypical role in adaptive immunity as DNA nucleases. Cas9 is typically guided to specific locations within the complex mammalian genome by a short RNA search string that has direct complementarity to a DNA target. However, some CRISPR-associated small RNAs have been demonstrated to serve as alternative guide RNAs that target endogenous transcripts instead of DNA. During the initial stage of the proposed work, the interactions of an RNA-regulating Cas9 complex with its endogenous target mRNA will be studied through biochemical reconstitution and characterization. Following heterologous expression in mammalian cells, computational modeling and systematic mutagenesis will be applied to understand the binding rules dictating ribonucleoprotein complex formation. This will inform reprogramming of the transcript targeting specificity. To exemplify the utility of these tools, the technology will be applied to study the role of long non-coding RNAs (lncRNAs) in the regulation of melanoma drug resistance. lncRNAs have diverse roles in regulating gene expression and have been implicated in both positive and negative regulation of cancer, yet little is known about their function in cancer drug resistance. To facilitate the discovery of novel therapeutic targets for combination melanoma therapy, a systematic pooled screen will be conducted to discover lncRNAs involved in melanoma resistance to BRAF-inhibitor therapy. The proposed platform technology development and high-throughput screening approach will enable the systematic discovery and functional interrogation of lncRNA-mediated biological processes, and can be generalized to study the biological mechanisms of transcriptional networks and therapeutic applications thereof.